Articular cartilage, an avascular tissue found at the ends of articulating bones, has no natural capacity to heal. During normal cartilage ontogeny, mesenchymal stem cells condense to form areas of high density and proceed through a series of developmental stages that ends in the mature chondrocyte. The final hyaline cartilage tissue contains only chondrocytes that are surrounded by a matrix composed of type II collagen, sulfated proteoglycans, and additional proteins. The matrix is heterogenous in structure and consists of three morphologically distinct zones: superficial, intermediate, and deep. Zones differ among collagen and proteoglycan distribution, calcification, orientation of collagen fibrils, and the positioning and alignment of chondrocytes (Archer et al., J. Anat. 189(1): 23-35, 1996; Morrison et al., J. Anat. 189(1): 9-22 1996, Mow et al., Biomaterials 13(2): 67-97, 1992). These properties provide the unique mechanical and physical parameters to hyaline cartilage tissue.
In 1965, a demineralized extraction from bovine long bones was found to induce endochondral bone formation in the rat subcutaneous assay (Urist Science 150: 893-899, 1965). Seven individual factors, termed Bone Morphogenetic Proteins (BMPs), were isolated to homogeneity and, because of significant sequence homology, classified as members of the TGFxcex2 super-family of proteins (Wozney, et al., Science 242: 1528-34, 1988; Wang et al., Proc. Nat. Acad. Sci. 87: 2220-2224, 1990). These individual, recombinantly-produced factors also induce ectopic bone formation in the rat model (Luyten et al., J. Biol. Chem. 264: 13377-80, 1989; Celeste et al., Proc. Nat. Acad. Sci. 87: 9843-50, 1990). In addition, in vitro tests have demonstrated that both BMP-2 and TGFxcex2-1 induce mesenchymal stem cells to form cartilage (Denker, et al., Differentiation 59(1): 25-34, 1995; Denker et al., 41st Ann. Orthop. Res. Society 465: 1995). Both BMP-7 and BMP-2 have been shown to enhance matrix production of chondrocytes in vitro (Flechtenmacher J. Arthritis Rheum. 39(11): 1896-904, 1996: Sailor et al., J. Orthop. Res. 14: 937-945, 1996). From these data we can conclude that not only are the BMPs important regulators of osteogenesis, but that they also play crucial roles during chondrogenic development in vitro.
A partially-purified protein mixture from bovine long bones, termed BP (Bone Protein), also induces cartilage and bone formation in the rat subcutaneous assay (Poser and Benedict, WO95/13767). BP in combination with calcium carbonate promotes bone formation in the body. In vitro, BP induces mesenchymal stem cells to differentiate specifically to the cartilage lineage, in high yields, and to late stages of maturation (Atkinson et al., J. Cellular Biochem. 65: 325-339, 1997).
The molecular mechanism for cartilage and bone formation has been partially elucidated. Both BMP and TGFxcex2 molecules bind to cell surface receptors (the BMP/TGFxcex2 receptors), which initiates a cascade of signals to the nucleus that promotes proliferation, differentiation to cartilage, and/or differentiation to bone (Massague Cell 85: 947-950, 1996).
In 1984, Urist described a substantially pure, but not recombinant BMP, combined with a biodegradable polylactic acid polymer delivery system for bone repair (U.S. Pat. No. 4,563,489). This system blends together equal quantities of BMP and polylactic acid (PLA) powder (100 xcexcg of each) and decreases the amount of BMP required to promote bone repair.
Hunziker (U.S. Pat. Nos. 5,368,858; 5,206,023) describes a cartilage repair composition consisting of a biodegradable matrix, a proliferation and/or chemotactic agent, and a transforming factor. A two stage approach is used where each component has a specific function over time. First, a specific concentration of proliferation/chemotactic agent fills the defect with repair cells. Secondly, a larger transforming factor concentration transforms repair cells into chondrocytes. Thereby the proliferation agent and the transforming agent may both be TGFxcex2 differing in concentration only. In addition, the patent discloses a liposome encapsulation method for delivering TFGxcex2-1 serving as transformation agent.
Hattersley et al. (WO 96/39170) disclose a two factor composition for inducing cartilaginous tissue formation using a cartilage formation-inducing protein and a cartilage maintenance inducing protein. Specific recombinant cartilage formation inducing protein(s) are specified as BMP-13, MP-52, and BMP-12, and cartilage maintenance-inducing protein(s) are specified as BMP-9. In one embodiment, BMP-9 is encapsulated in a resorbable polymer system and delivered to coincide with the presence of cartilage formation inducing protein(s).
Laurencin et al., (U.S. Pat. No. 5,629,009) disclose a chondrogenesis-inducing device, consisting of a polyanhydride and polyorthoester, that delivers water soluble proteins derived from demineralized bone matrix, TGFxcex2, EGF, FGF, or PDGF.
The results of the approaches to cartilage repair as cited above are encouraging but they are not satisfactory. In particular, the repair tissue arrived at is not fully hyaline in appearance and/or it does not contain the proper chondrocyte organization. Furthermore, previous approaches to cartilage repair have been addressed to very small defects and have not been able to solve problems associated with repair of large, clinically relevant defects.
One reason that previous approaches failed to adequately repair cartilage may be that they were not able to recapitulate natural cartilage ontogeny faithfully enough, this natural ontogeny being based on a very complicated system of different factors, factor combinations and factor concentrations with temporal and local gradients. A single recombinant growth factor or two recombinant growth factors may lack the inductive complexity to mimic cartilage development to a sufficient degree and/or the delivery systems used may not have been able to mimic the gradient complexity of the natural system to a satisfactory degree.
Previous approaches may also have failed because growth factor concentrations were not able to be maintained over a sufficient amount of time, which would prevent a full and permanent differentiation of precursor cells to chondrocytes. The loss of growth factor could be caused by diffusion, degradation, or by cellular internalization that bypasses the BMP/TGFxcex2 receptors. Maintaining a sufficient growth factor concentration becomes particularly important in repair of large sized defects that may take several days or several weeks to fully repopulate with cells.
The object of this invention is to create a composition for improved cartilage repair in vivo. The inventive composition is to enable in vivo formation of repair cartilage tissue which tissue resembles endogenous cartilage (in the case of articular cartilage with its specific chondrocyte spatial organization and superficial, intermediate, and deep cartilage zones) more closely than repair tissue achieved using known compositions for inducing cartilage repair. A further object of the invention is to create a device for cartilage repair which device contains the inventive composition.
This object is achieved by the composition and the device as defined by the claims.
The inventive composition basically consists of a naturally derived osteoinductive and/or chondroinductive mixture of factors (e.g. derived from bone) or of a synthetic mimic of such a mixture combined with a nanosphere delivery system. A preferred mixture of factors is the combination of factors isolated from bone, known as BP and described by Poser and Benedict (WO 95/13767). The nanosphere delivery system consists of nanospheres defined as polymer particles of less than 1000 nm in diameter (whereby the majority of particles preferably ranges between 200-400 nm) in which nanospheres the combination of factors is encapsulated. The nanospheres are loaded with the mixture of factors in a weight ratio of 0.001 to 17% (w/w), preferably of 1 to 4% (w/w) and have an analytically defined release profile (see description regarding FIG. 2) showing an initial burst of 10 to 20% of the total load over the first 24 hours and a long time release of at least 0.1 per day during at least seven following days, preferably of 0.1 to 1% over the following 40 to 60 days. The nanospheres are composed of e.g. (lactic acid-glycolic acid)-copolymers (Poly-(D,L)lactic acid-glycolic acid) made of 20 to 80% lactic acid and 80 to 20% of glycolic acid, more preferably of 50% lactic acid and 50% of glycolic acid.
The loaded nanospheres are e.g. made by phase inversion according to Mathiowitz et al. (Nature, 386: 410-413, 1997) or by other methods known to those skilled in the art (Landry, Ph.D Thesis, Frankfurt, Germany).
The inventive composition is advantageously utilized as a device comprising any biodegradable matrix including collagen type I and II, and hyaluronic acid in which matrix the nanospheres loaded with the factor combination is contained. The matrix can be in the form of a sponge, membrane, film or gel. The matrix should be easily digestible by migrating cells, should be of a porous nature to enhance cell migration, and/or should be able to completely fill the defect area without any gaps.
It is surprisingly found that the inventive composition consisting of an osteoinductive and/or chondroinductive combination of factors (e.g. derived from natural tissue) encapsulated in nanospheres as specified above, if applied to a defect area of an articular cartilage, leads to the transformation of virtually all precursor cells recruited to the repair area to chondrocytes, and furthermore, leads to a homogenous chondrocyte population of the repair area and to a chondrocyte order and anisotropic appearance as observed in endogenous hyaline cartilage. These findings encourage the prospect that the inventive composition may lead to significant improvements also regarding repair of large defects.
As mentioned above, instead of an osteoinductive and/or chondroinductive mixture of factors derived from bone (BP), the inventive composition may comprise natural factor mixtures derived from other tissues (e.g. cartilage, tendon, meniscus or ligament) or may even be a synthetic mimic of such a mixture having an osteoinductive and/or chondroinductive effect. Effective mixtures isolated from natural tissue seem to contain a combination of proliferation, differentiation, and spatial organizing proteins which in combination enhance the tissue rebuilding capacity more effectively than single proteins (e.g. recombinant proteins).
The specified, analytically defined release profile of such factor mixtures from nanospheres results in the formation of concentration gradients of proliferation and differentiation factors, which obviously mimics the complex gradients of factors observed during natural development very well. The nanosphere extended release profile is sufficient to provide growth factor during the time frame that repair cells arrive into the matrix. The release profile obviously leads to a homogenous population of a matrix with precursor cells, to full differentiation of virtually all of the precursor cells to chondrocytes, and to the formation of an endogenous hyaline cartilage structure.
Another advantage of the inventive composition is that when the nanospheres are placed in a matrix to form a device for cartilage repair, they are randomly distributed and remain in place when in a joint cartilage defect. During cellular infiltration and differentiation, the nanospheres are in the correct position over the correct time frame.
Nanospheres have been demonstrated to adhere to the gastrointestinal mucus and cellular linings after oral ingestion (Mathiowitz et al., Nature, 386 410-413 1997). We envisage that nanospheres also adhere to cartilage precursor cells and furthermore, may also adhere to BMP/TGFxcex2 receptors located on the cell membrane. This property allows localized high-efficiency delivery to the target cells and/or receptors. Because of the nanosphere small size and the chemical properties, they are more effective than liposomes or diffusion controlled delivery systems. The efficient delivery to the receptors will facilitate chondrogenesis.
Derived from the above findings, we envisage the following mechanism for cartilage repair using the effect of the inventive composition. During the first 24 hours (initial burst) 10 to 20% of the total load of the factor mixture is released from the nanospheres into the matrix and diffuses into the synovial environment. Following the initial burst, the nanospheres begin to release factors at a slow rate, which produces gradients of proliferation, differentiation, and spatial organizing proteins. In response to such gradients, precursor cells migrate to the defect site. The loaded nanospheres adhere to cartilage precursor cells and to the BMP and TGFxcex2 receptors to provide localized highly efficient delivery. The precursor cells become differentiated to chondrocytes and secrete type II collagen and cartilage-specific proteoglycans. The composition of the present invention stimulates differentiation of virtually all of these cells to overt chondrocytes and induces an ordered cartilage structure which closely resembles hyaline cartilage. Furthermore, we envisage that this release system will allow homogenous repair of large defect sites and repair of defects from patients with low quantities of precursor cells.
For in vivo cartilage repair, the inventive device consisting of a matrix and the loaded nanospheres is placed in a chondral lesion that was caused by trauma, arthritis, congenital, or other origin. The damage can result in holes or crevices or can consist of soft, dying, or sick cartilage tissue that is removed surgically prior to implantation of the device. Because of the unique properties of the inventive device precursor cells populate the matrix, differentiate to chondrocytes, and form hyaline cartilage.
Application of the inventive composition (without matrix) e.g. by injection can be envisaged also, in particular in the case of small defects. Thereby at least 2 xcexcg of the composition per ml of defect size is applied or at least 20 ng of the osteoinductive and/or chondroinductive mixture encapsulated in the nanospheres is applied per ml defect size.
The inventive composition and the inventive device are suitable for repair of cartilage tissue in general, in particular for articular cartilage and for meniscus cartilage.